Enzymes that can catalyze the breakdown of oxalic acid have potential therapeutic application in the treatment of human pathological conditions associated with the accumulation of this compound in the blood and/or urine. This proposal outlines the continuation of integrated experimental and computational studies aimed at understanding the fundamental biochemistry and regulation of oxalate decarboxylase (OxDC), an enzyme that catalyzes the conversion of oxalate to carbon dioxide and formate. The goals of this project are (i) to test mechanistic proposals for the involvement of radical species in the molecular processes that result in cleavage of the oxalate C-C bond, (ii) to investigate the role of dioxygen and higher oxidation states of the active site manganese ion in the catalytic mechanism, and (iii) to evaluate changes in local protein environment function so as to modulate the intrinsic reactivity of the Mn(II) center in the enzyme. An integrated experimental and computational strategy, using techniques in bioinorganic chemistry, molecular spectroscopy, enzyme kinetics and protein engineering, X-ray crystallography, and computational chemistry, will be pursued in these efforts, which have two major specific aims. In the first aim, biophysical structural methods, EPR spectroscopy and density functional theory (DFT) calculations will be used to validate the hypothetical mechanism of OxDC-catalyzed decarboxylation, and establish the precise manganese oxidation state that mediates the reaction. The second aim will focus on the kinetic and biophysical characterization of OxDC mutants in an effort to evaluate the effect of protein environment in modulating the chemical reactivity of the Mn(II) center(s) in the enzyme. In addition to the general biochemical implications of these efforts for our knowledge of the chemical mechanisms by which enzymes can catalyze difficult reactions, and the molecular processes that seem to be used in evolving proteins with altered catalytic properties, these efforts should provide a platform for long-term translational research on the development of novel therapies for the clinical treatment and/or prevention of oxalate-related disease.